SPECIFIC GRP78 EXPRESSION-INHIBITION RNAi SEQUENCE, MEDICINE THEREOF AND METHOD THEREOF

ABSTRACT

The present invention discloses a specific GRP78 expression-inhibition RNAi sequence, a medicine thereof and a method thereof, wherein an RNAi sequence 5′-AAGGATGGTTAATGATGCTGAGAA-3′ [SEQ. ID NO: 1] complementary to GRP78 forms a special hair-pin structure inside cancer cells to specifically and effectively inhibit GRP78 expression and then inhibit the canceration process, including the growth, migration, invasion, and metastasis of cancer.

The current application is a divisional application of and claims apriority to the U.S. application Ser. No. 12/174,997, filed on Jul. 17,2008, which claims a priority to a foreign application, Taiwan 97101260,filed on Jan. 11, 2008.

The current application claims the Petition To Accept Color Drawingsfiled on Jul. 17, 2008 with the U.S. Ser. No. 12/174,997.

The current application claims the Sequence Listing filed on Oct. 31,2008 and the Computer Readable Form filed on Nov. 28, 2008 with the U.S.Ser. No. 12/174,997.

All references to the claimed priorities, color drawings, sequencelisting, and the computer readable form are incorporated herewith in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an RNA interference technology,particularly to a specific GRP78 expression-inhibition RNAi sequence, amedicine thereof and a method thereof.

2. Description of the Related Art

The 78-kDA glucose regulated protein (GRP78), also known as hsp70-5,hspA5 or Bip, is one member of the heat shock protein 70 (HSP70) family.GRP78 is a functional protein, which implements folding up anewly-synthesized protein to have an appropriate conformation inendoplasmic reticulum. According to previous researches, a crisis ofcells, such as a hypoxia state or an ultraviolet radiation, will triggerGRP78 to assist in the degradation of the incorrectly folded protein.Therefore, GRP78 is thought to be a stress sensor of endoplasmicreticulum, which functions as the cyto-protection and anti-apoptosismechanisms of cells.

It is also found in some researches that GRP78 can function as thelabeled protein of breast cancer. GRP78 can help cells survive in aglucose-deficiency environment. Some researches show: a patient havingoverexpressed GRP78 has a higher recurrence rate no matter in what stageof cancer, and GRP78 overexpression makes cells of breast cancer have ahigher chemotherapy resistance. This finding gives physicians a veryimportant therapeutic reference. If GRP78 concentration is tested beforechemotherapy, unnecessary medicine and useless therapy can be avoided.Besides, chemotherapy sensitivity can be promoted via reducing GRP78expression.

Further, the Inventor found that GRP78 expression in head and neckcancer cells is much higher than the expression in non-cancer cells andthat GRP78 expression correlates with clinical malignant indications,such as tumor size, tumor depth, lymph metastasis. Therefore, GRP78expression can be used as a reference for tumor grading. However, in thecurrent cancer therapeutic technology, neither molecular inhibitiontechnology nor molecular therapeutic medicine is designed to effectivelyinhibit the expression of GRP78—the overexpressed gene in breastcancer/head and neck cancer.

SUMMARY OF THE INVENTION

The primary objective of the present invention is to provide a specificGRP78 expression-inhibition RNAi sequence, a medicine thereof and amethod thereof, which is based on the fact that a GRP78-related RNAisequence can effectively inhibit GRP78 expression, and whereby thegrowth, metastasis and invasion of cancer cells can be inhibited.

To achieve the abovementioned objective, the present invention disclosesa specific GRP78 expression-inhibition RNAi sequence, a medicine thereofand a method thereof, wherein an RNA interference technology is used toinhibit GRP78 expression. In the RNA interference technology, a dicerprotein recognizes a small segment of RNA having 18-24 nucleotides,which matches a messenger RNA (mRNA) inside cells, and cuts off the mRNAto inhibit the expression of a special gene. Such a method canspecifically inhibit the expression of a special gene. The inhibitioneffect of the RNA interference technology closely correlates with thesmall segment of sequence, and the Inventor has found a specificsequence in GRP78, which can more effectively inhibit GRP78 expressionthan other sequences.

The RNA interference sequence of the present invention is5′-AAGGATGGTTAATGATGCTGAGAA-3′ (SEQ ID NO: 1), which is a sequence of 24nucleotides beginning from Position 1821 of GRP78 mRNA and able tofunction as an RNA interference mechanism for GRP78 molecules insidecells. The RNA interference sequence for GRP78 can be applied tocancer-inhibition medicines. The medicine contains a plasmid carryingthe RNAi sequence and can be sent into the body of a cancer patient toinhibit the growth, metastasis and invasion of cancer cells.

Below, detailed description, in cooperation with the drawings, is usedto further demonstrate the objectives, characteristics and efficacies ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIG. 1 is a diagram schematically showing a structure formed insidecells by a specific GRP78 expression-inhibition RNAi sequence accordingto the present invention.

FIGS. 2( a)˜10 show experimental data according to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The specific GRP78 expression-inhibition RNAi sequence designed by theInventor is a sequence of 24 nucleotides 5′-AAGGATGGTTAATGATGCTGAGAA-3′,(SEQ ID NO: 1) which begins from Position 1821 of GRP78 mRNA. The RNAisequence of the present invention can effectively inhibit the expressionof GRP78 gene and thus can inhibit the growth of cancer cells, whereinthe cancer cells include breast cancer cells and head and neck cancercells. The theory and efficacies of the present invention will beverified with experiments on head and neck cancer cells and the analysesthereof.

The processes of the experiments are described in detailed below:

-   (1) Synthesizing the GRP78 expression-inhibition RNAi sequences of    the present invention and the control sequences (scramble    sequences), and then synthesizing the plasmids carrying the    abovementioned sequences:    -   The GRP78-RNAi sequences are        5′-AAGGATGGTTAATGATGCTGAGAAgaagcttgTTCTCAGCATC ATTAACCATCCTT-3′        (SEQ ID NO: 2), and        5′-GGTTAATGATGCTGAGAActtcgaacTTCTCAGCATCATTAACC-3′ (SEQ ID NO:        3).    -   The GRP78-scramble RNA sequences are        5′-AAGGATAATGATGCTGAGGGTTAAgaagcttgTTAACCCTCAG CATCATTATCCTT-3′        (SEQ ID NO: 4), and        5′-AATGATGCTGAGGGTTAActtcgaacTTAACCCTCAGCATCATT-3′ (SEQ ID NO:        5).    -   The boldface uppercases is the restriction enzyme site cloned to        the plasmid, and the lowercases is the hair-pin structure.-   (2) Connecting the synthesized sequences to the expression vectors:    -   Two strands of the synthesized nucleotides are respectively        heated to 95° C. for 10 minutes and then cooled to the ambient        temperature. Thus, the synthesized sequences can match to form a        two-strand GRP78-RNAi structure, wherein the GRP78-RNAi        sequences are 5′-AAGGATGGTTAATGATGCTGAGAAgaagcttgTTCTCAGCATC        ATTAACCATCCTT-3′ (SEQ ID NO: 2), and        5′-GGTTAATGATGCTGAGAActtcgaacTTCTCAGCATCATTAACC-3′ (SEQ ID NO:        3), and wherein the GRP78-scramble sequences are        5′-AAGGATAATGATGCTGAGGGTTAAgaagcttgTTAACCCTCAG CATCATTATCCTT-3′        (SEQ ID NO: 4), and        5′-AATGATGCTGAGGGTTAActtcgaacTTAACCCTCAGCATCATT-3′ (SEQ ID NO:        5).    -   The vector is a self-manufactured pTOPO-U6 (SEQ ID NO: 6),        wherein U6 promoter is cloned to a pTOPO vector. After the        hair-pin structure interference sequence is formed, the EcoRV        and BbsI portions of a restriction enzyme are used to dissect        the expression vector, and two strands of RNAi sequences are        respectively embedded into the expression vector. Next, a T4 DNA        ligase is used to join them together to form a GRP78-RNAi        plasmid or a GRP78-scramble plasmid. Next, the joined sequences        are sent into bacteria E. coli and expressed therein. Then, the        plasmid carrying GRP78-RNAi or GRP78-scramble is extracted.-   (3) Proving that the interference sequence of the present invention    is indeed able to inhibit GRP-78 expression:    -   With the plasmid of TOPO-U6 being the vector, one experimental        group and two control groups are prepared: Control group I has a        plasmid expression vector and is named the vector control group;        Control group II has a recombination GRP78-RNAi sequence with        none inhibition effect and is named the scramble group; the        experimental group has an interference sequence plasmid and is        named the GRP78-RNAi group. They are respectively transfected        into head and neck cancer cell strains, such as nasopharyngeal        cancer cell lines (NPC-BM1, NPC-BM2 and NPC-076), an oral cancer        cell line (OECM1), laryngeal cancer cell lines (FADU and Detroit        562). After the intracellular transcript process, the GRP78-RNAi        plasmid will form a special hair-pin structure inside cells, as        shown in FIG. 1.    -   Two days later, the cells are collected, and protein is        extracted therefrom. A western blot analysis method is used to        compare the GRP78 expressions in the three groups. Refer to FIG.        2( a)˜FIG. 2( f). Compared with the vector control group, the        interference sequence of the GRP78-RNAi group can obviously        inhibit GRP78 expression. In the experiments, Actin expression        is used in protein quantification, but GRP78-RNAi does not        affect the expression of Actin. The GRP78-RNAi sequence of the        present invention is proved to be able to specifically and        effectively inhibit GRP78 expression.-   (4) Proving that the inhibition of GRP78 expression by the present    invention is indeed able to inhibit the growth, metastasis and    invasion of head and neck cancer cells:    -   a. Proving that inhibiting GRP78 expression can inhibit the        growth and colony formation of head and neck cancer cells:        -   The expression variation of the three groups in cell facet            is used to investigate the effects of the GRP78-RNAi method            of the present invention. The growth of head and neck cancer            cells within 1-5 days is investigated with cell count and            colony formation analysis. Refer to FIG. 3( a)˜FIG. 3( f).            From cell count, it is shown: inhibiting GRP78 expression            can inhibit the growth of cancer cells in all six cell            strains Refer to FIG. 4( a)˜FIG. 4( f). Crystal violet is            used to stain cells and quantify the number of cell            colonies. From colony formation analysis, it is shown: the            size and number of cell colony in the experimental group is            smaller that that in the control groups. From the above            mentioned two test methods, it is proved that the specific            GRP78 expression-inhibition RNAi sequence of the present            invention can indeed inhibit the growth of head and neck            cancer cells.    -   b. Proving that inhibiting GRP78 expression can inhibit the        migration and invasion of head and neck cancer cells:        -   In addition to the influence of the specific GRP78            expression-inhibition RNAi sequence on the growth of head            and neck cancer cells, the influence thereof on the            migration and invasion of cancer cells is also investigated            herein. A wound healing assay is used to investigate the            influence of the GRP78 expression-inhibition RNAi sequence            on the mobility of head and neck cancer cells. Head and neck            cancer cells are planted on the bottom of culture dishes,            and a sharpened plastic tip is used to generate artificial            wounds. Refer to FIG. 5( a)˜FIG. 5( f). From analyzing the            healing capability of the artificial wounds and the cell            mobility, it is proved that inhibiting GRP78 expression can            inhibit the mobility of head and neck cancer cells. A            Transwell invasion assay is used to investigate the invasion            capability of cancer cells, wherein on the upper layer of            the Transwell has a layer of Matrigel. If the cancer cells            are aggressive invasion, they will digest Matrigel and            invade from the upper chamber of Transwell to the lower            chamber. From FIG. 6( a)˜FIG. 6( f), it is proved that the            specific GRP78 expression-inhibition RNAi sequence can            indeed inhibit the invasion of head and neck cancer cells.    -   c. Proving with an in vivo method that inhibiting GRP78        expression can inhibit the growth of cancer cells:        -   Five-week-old BALB/C nude mice are used in the in vivo            experiment. Total of 10⁷ of FADU and Detroit 562 laryngeal            cancer cells are subcutaneously implanted in the nude mice.            Three days later, 50 μg of the control-group vectors or 50            μg of the GRP78-RNAi plasmid (vectors carrying the            GRP78-RNAi sequence) are respectively injected into the nude            mice from the caudal veins thereof. Then, two doses of 25 μg            GRP78-RNAi plasmid or vectors are further injected into the            nude mice each week. Each group has eight mice, and the            duration of the experiments is eight weeks. The lengths,            widths and heights of cancers are measured to calculate the            volumes of cancers and monitor the growth states of cancers.            Refer to FIG. 7( a)˜FIG. 7( b). In the experimental group,            GRP78 expression is inhibited, and the xenografted cancer            cells grow slowly. In the control groups, cancer cells grow            persistently. In the experiment of FADU cancer cells, the            cancer is 44% inhibited (P=0.003) in the eighteenth day, and            69% inhibited (P=0.004) in the thirty-fourth day. In the            experiment of Detroit cancer cells, the cancer is 60%            inhibited (P=0.017) in the eighteenth day, and 66% inhibited            (P=0.016) in the thirty-ninth day. Six weeks later, the            cancer tissues are taken off from the nude mice, and an            immunohistochemistry method is used to analyze GRP78            expression in cancer tissues. Refer to FIG. 8( a)˜FIG. 8(            b). It is proved from the test on the cancer sections that            GRP78 expression is inhibited by the RANi sequence of the            present invention. The experiment proves that inhibiting            GRP78 expression can indeed inhibit the growth of in vivo            cancer cells.    -   d. Proving with an in vivo method that inhibiting GRP78        expression can inhibit the metastasis of cancer cells:        -   The IVIS system (In vivo imaging system) was used to monitor            metastatic potential using Fadu xenograft mice model. First,            the Fadu cell line stably transfected with luciferase gene            was established. The Fadu cells were injected with 5×105            cells through the tail vein. Three days after tumor cell            xenografting, the mice were randomly divided into 3 groups            of 7 mice each. The experimental group was injected            intravenously with 50 μg of Grp78-RNAi plasmid in 50 μl PBS,            followed by a booster of 25 μg of the plasmid in 25 μl PBS            twice a week for 3 weeks. The two control groups were            injected on the same schedule as the experimental group but            with either vector or scramble plasmids.        -   For IVIS examination, luciferin substrate (100 μl of 30            mg/ml in PBS) was injected subcutaneously. After 10 min,            mice were anesthetized with an isoflurane-oxygen mixture.            Photoemissions from the luciferin-luciferase reaction were            detected with a sensitive CCD camera. The imaging system            first produced a photographic image in the chamber under dim            illumination, followed by luminescent image acquisition. The            overlay of the pseudocolor images represents the spatial            distribution of photo counts produced by active luciferase.            Living Image software (Xenogen) was used to integrate the            bioluminescence signals and measure photo flux obtained from            the mice. Refer to FIG. 9. The lucerin flux in the group of            RNAi treated mice was significantly lower than in            scramble-plasmid treated controls. Quantitation for the            lucerin flux showed average reductions to 8.8% at day 11 and            7.0% at day 20.        -   The mice were then sacrificed and the livers were examined            to evaluate the metastatic potential of the tumor            xenografts. Refer to FIG. 10. Examination on the livers of            the RNAi treated mice also demonstrated that Grp78 knockdown            suppressed tumor metastasized to the organs. Six of the 7            mice in scramble-treated groups had one or more liver tumor            mass, whereas none of tumor was found in the livers of RNAi            treated group.

In conclusion, the present invention discloses a specific GRP78expression-inhibition RNAi sequence, a medicine thereof and a methodthereof, wherein an RNA interference technology is used to specificallyand effectively inhibit intracellular GRP78 expression and then inhibitthe canceration process, including the growth, migration, invasion andmetastasis of cancer. The present invention can specifically suppressGRP78 expression in the high level of cancer cells, thus, reduce damageof non-cancer cells and decrease the side-effect of chemotherapy.

The embodiments described above are only to exemplify the presentinvention but not to limit the scope of the present invention. Anyequivalent modification or variation according to the spirit of thepresent invention is to be also included within the scope of the presentinvention, which is based on the claims stated below.

1. A specific 78-kDA glucose regulated protein (GRP78)expression-inhibition RNA interference (RNAi) sequence5′-AAGGATGGTTAATGATGCTGAGAA-3′ [SEQ. ID NO: 1], which is used to inhibitgrowth, metastasis and invasion of cancer cells.
 2. The specific 78-kDAglucose regulated protein expression-inhibition RNA interferencesequence of claim 1, wherein said cancer cells are head and neck cancercells.
 3. The specific 78-kDA glucose regulated proteinexpression-inhibition RNA interference sequence of claim 2, wherein saidhead and neck cancer cells include nasopharyngeal cancer cells.
 4. Amedicine for specifically inhibiting 78-kDA glucose regulated protein(GRP78) expression, which contains a plasmid carrying an RNAinterference (RNAi) sequence and is used to inhibit growth, migration,invasion, and metastasis of cancer cells, wherein said RNA interferencesequence is 5′-AAGGATGGTTAATGATGCTGAGAA-3′ [SEQ. ID NO: 1].
 5. Themedicine for specifically inhibiting 78-kDA glucose regulated proteinexpression of claim 4, wherein said cancer cells are head and neckcancer cells.
 6. The medicine for specifically inhibiting 78-kDA glucoseregulated protein expression of claim 5, wherein said head and neckcancer cells include nasopharyngeal cancer cells.